INTRODUCTION

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VASOCONSTRICTION is often associated with depolarization of the smooth muscle membrane potential and increased calcium influx through voltage-dependent calcium channels (18). This is particularly evident in resistance arteries, which depolarize in response to various stimuli, including increased intravascular pressure (11). Vasoconstriction caused by elevation of intravascular pressure is referred to as myogenic tone. Although the central role of voltage-dependent calcium channels in myogenic tone and the nature of negative feedback regulation of this response are well known (17), the ionic and molecular mechanisms of depolarization associated with myogenic tone are still being resolved. Welsh et al. (25) have recently provided evidence of a major role for nonselective cation channels in this response in cerebrovascular smooth muscle cells. These channels are activated by cell swelling and by increased intravascular pressure and are similar to cation channels present in other types of arterial smooth muscle (12, 22, 28). Moreover, our studies indicate that transient receptor potential channels, first described in the Drosophila retina (15) and now known to be present in most mammalian tissues (1), are molecular candidates for these cation channels in cerebrovascular smooth muscle cells (25).

In the present study, to further characterize the cellular mechanisms associated with pressure-induced depolarization of vascular smooth muscle cells, we examined the possible involvement of signaling molecules associated with activation of phospholipase C (PLC), specifically diacylglycerol (DAG) and protein kinase C (PKC). This approach was based on evidence indicating a prominent role of this signaling pathway in activation of myogenic tone in resistance arteries (6) and the apparent involvement of PLC and its products in regulation of transient receptor potential channels (1). Our data indicate a significant role for PKC in activation of nonselective cation channels in cerebrovascular myocytes and in the pressure-induced depolarization of smooth muscle cells in intact cerebral arteries.

	METHODS

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Cell preparation. Cerebellar and posterior cerebral arteries were removed from 12- to 16-wk-old Sprague-Dawley rats (Charles River Laboratories; St. Constant, Quebec, Canada) after euthanasia by lethal dose of pentobarbital sodium and exsanguination. To isolate myocytes, vessels were cleaned of connective tissue, cut into 2-mm segments, and placed in the following cell isolation solution (in mM): 60 NaCl, 80 Na glutamate, 5 KCl, 2 MgCl₂, 10 glucose, and 10 HEPES; pH 7.2. The segments were incubated at 37°C in 1 mg/ml papain and 1 mg/ml dithioerythritol for 20-30 min, followed by 10 min incubation in 0.5 mg/ml type F collagenase and 1.0 mg/ml hyaluronidase. They were then washed three times in ice-cold bath solution and triturated to release myocytes. Cells were stored on ice in isolation solution for use the same day.

Patch clamp recording. Conventional whole cell patch clamp was used to record ionic currents. Recording electrodes (resistance, 4-7 M) were pulled from borosilicate glass (1.5 mm OD, 1.17 mm ID; Sutter Instrument, Novato, CA) and coated with wax to reduce capacitance. Cells were voltage clamped and held at 50 mV between ramp protocols; whole cell currents were recorded during voltage ramp from 100 to 100 mV (100 mV/s). Voltage ramp recordings shown represent the average of five sweeps. Membrane currents were filtered at 1 kHz, digitized at 5 kHz, and stored for subsequent analysis. pCLAMP 8.1 and Clampfit 8.1 (Axon Instruments) were used for data acquisition and analysis. Quantification of whole cell currents was done at 100 mV. Cell capacitances ranged from 21 to 15 pF and were measured with cancellation circuitry in the voltage-clamp amplifier (Axopatch 200A amplifier, Axon Instruments). All recordings were performed at room temperature (22°C).

Solutions. Control recordings were made in the following isotonic bath solution (in mM): 120 NaCl, 10 glucose, 10 HEPES, 0.010 CaCl₂ (pH 7.2), and sufficient mannitol to bring osmolarity to 300 mosM. Hypotonic solution had identical ionic concentrations, with sufficient mannitol to bring osmolarity to 250 mosM. Bath N-methyl-D-glucamine (NMDG) solution contained (in mM) 120 NMDG, 10 glucose, 10 HEPES, and 0.010 CaCl₂, pH 7.2 (~120 mM HCl). Pipette solution was (in mM) 120 NMDG, 120 aspartic acid, 5 HEPES, 5 EGTA, 1 disodium-ATP, and 5 NaCl, pH 7.2, with sufficient mannitol added to bring osmolarity to 300 mosM. Chemicals were obtained from Sigma Chemical (St. Louis, MO) and Cabiochem (San Diego, CA).

Membrane potential measurements. Segments of the superior cerebellar and posterior cerebral artery (~150 µm in diameter, 2-3 mm in length) were removed from the brain and placed in physiological salt solution (PSS) of the following composition (in mM): 119 NaCl, 3 KCl, 1.7 KH₂PO₄, 1.2 MgSO₄, 25 NaHCO₃, 0.02 EDTA, 1.6 CaCl₂, and 11 glucose, pH 7.4. Endothelial cells were removed from all arteries to assure that effects of various agents used in the studies could be attributed to actions on the smooth muscle cells. Removal of the endothelial lining was accomplished by passing air bubbles through the lumen of the cannulated artery for 2 min followed by perfusion with PSS. For measurement of membrane potential, arteries were then mounted in a myograph chamber (Living Systems Instruments; Burlington, VT). The arteries were continuously superfused with warmed (37°C) PSS that was gassed with 95% oxygen-5% CO₂ (pH 7.4). Membrane potential was measured by inserting a glass microelectrode filled with 0.5 M KCl (tip resistance, 120-180 M) into the vessel wall. The criteria for a successful cell penetration were 1) a sharp negative membrane potential deflection on entry; 2) a stable potential for at least 1 min after entry; and 3) a sharp positive membrane potential deflection on removal.

	RESULTS

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A basal cation current similar to that previously identified in these cells (25, 26) was identified in freshly isolated cerebral arterial myocytes. Bath application of the membrane-permeant DAG analog 1,2-dioctanoyl-sn-glycerol (DOG, 100 µM) stimulated the steady-state inward current at 60 mV (Fig. 1A). Replacement of the external Na⁺ with the nonconducting NMDG⁺ greatly diminished this current. In voltage ramp experiments, DOG stimulated the inward current (Fig. 1B). The average stimulation of this current at 100 mV was 232 ± 43% of the control. This current was also stimulated by 100 µM 1-oleoyl-2-acetyl-sn-glycerol, a different membrane-permeant DAG analog, to 154 ± 16% (n = 3) of control.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Protein kinase C (PKC) inhibitors block a 1,2-dioctanoyl-sn-glycerol (DOG)-activated cation current in cerebrovascular myocytes A: DOG stimulates a cation current in a cell held at 60 mV. Isotonic solution was replaced with isotonic solution containing DOG (100 µM) where noted. NMDG, N-methyl-D-glucamine B: DOG stimulated the inward current under voltage ramp conditions. C: chelerythrine (3 µM) blocks the response to DOG. D: calphostin C (0.5 µM) blocks the response to DOG. E: intracellular PKC inhibitor (PKI) peptide PKC 19-31 (1 µM) blocks the response to DOG. F: summary of effects at 100 mV (n: DOG = 5, chelerythrine = 5, calphostin = 3, PKI = 3). * Significant difference from control, P < 0.05.

The activation of the inward cation current by DOG was blocked by three inhibitors of PKC: chelerythrine (3 µM, Fig. 1C), calphostin C (0.5 µM, Fig. 1D), or pipette application of the peptide PKC 19-31 (10 µM, Fig. 1E). A summary of these effects is shown in Fig. 1F.

Activation of this current could also be produced by exposure of cells to the DAG lipase inhibitor RCH-80267 (50 nM) (Fig. 2A, 140 ± 8.3% of control). This suggests that there is a tonic production of DAG in the basal state and that the basal concentration of DAG is augmented by inhibition of its breakdown. In addition, the PKC activator phorbol myristate acetate (PMA, 1 µM) caused significant activation of the current (Fig. 2B, 209 ± 20% of control), suggesting modification of the current by PKC.

View larger version (8K): [in this window] [in a new window]   Fig. 2.   RCH-80267 and phorbol myristate acetate (PMA) stimulate cation currents in cerebrovascular myocytes. A: diacylglycerol (DAG) lipase inhibitor RHC-80267 (50 nM) activates the inward current. B: phorbol ester PMA (1 µM) activates inward current. C: summary of results (n: 5 for RCH-80267 and 7 for PMA). * Significant difference from control, P < 0.05.

Replacing the bath media with hypotonic media caused the cells to swell and stimulated a cation current (Fig. 3A). In voltage ramp experiments, the inward current at 100 mV was increased an average of 229 ± 15% of control by hypotonic swelling (Fig. 3B). Bath application of chelerythrine (3 µM, Fig. 3C) or calphostin C (0.5 µM, Fig. 3D), as well as pipette application of PKC 19-31 (1 µM, Fig. 3E), inhibited the activation of this current. A summary of these effects can be seen in Fig. 3F.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   PKC inhibitors block a swelling-activated cation current in cerebrovascular myocytes. A: hypotonic swelling stimulates a cation current in a cell held at 80 mV. Isotonic solution was replaced with 250 mosM solution (hypotonic) where noted. B: hypotonic swelling stimulates the inward current under voltage ramp conditions. C: chelerythrine (3 µM) blocks the response to hypotonic solution. D: calphostin C (0.5 µM) blocks the response to hypotonic solution. E: intracellular PKC inhibitor peptide PKC 19-31 (1 µM) blocks the response to hypotonic solution. F: summary of effects at 100 mV (n: hypotonic = 12, chelerythrine = 4, calphostin C = 5, PKI = 5). * Significant difference from control, P < 0.05.

We then tested the effects of DOG, PMA, and the PKC inhibitor chelerythrine on smooth muscle membrane potential in intact pressurized arteries. In arteries held at 20 mmHg, a pressure at which little myogenic depolarization is present (11), DOG and PMA caused substantial smooth muscle depolarization (Fig. 4, A and B). These effects were fully reversed by chelerythrine. The smooth muscle membrane potential of arteries pressurized to 60 mmHg was depolarized by about 16 mV compared with membrane potentials at 20 mmHg. Addition of chelerythrine to these arteries hyperpolarized the membrane potential by 15 mV (Fig. 4C). These results indicate that PKC activation can cause a substantial depolarization of smooth muscle that is similar in magnitude to that caused by elevation of intravascular pressure. The inhibitory effect of chelerythrine on the pressure-induced depolarization suggests a central role for PKC in this response.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   PKC inhibitor chelerythrine reverses the depolarizing effects of DOG, PMA, and increased pressure in intact cerebral arteries. A: effect of DOG (100 µM) on membrane potential (20 mmHg intravascular pressure) and block of this effect by chelerythrine (3 µM). B: effect of PMA (1 µM) on membrane potential (20 mmHg intravascular pressure) and block by chelerythrine. C: effect of chelerythrine (3 µM) on resting membrane potential at 60 mmHg intravascular pressure. * Significant difference from control, P < 0.05.

	DISCUSSION

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In this study we have shown that a DAG analog activates a cation current in cerebrovascular smooth muscle cells. This current is similar to nonselective cation currents activated by cell swelling that were previously implicated in the mechanism of depolarization induced by elevation of intravascular pressure (26). In addition, PKC is involved in modulation of this smooth muscle cation current, because the currents activated by DAG and cell swelling were eliminated when PKC was inhibited. Also, PMA, a direct activator of PKC, increased the cation currents. PKC blockers reversed smooth muscle depolarization induced by elevation of intravascular pressure in intact arteries. This directly implicates a role for PKC in regulation of a key pathway associated with the development of myogenic tone in cerebral arteries, i.e., pressure-induced depolarization, involving, at least in part, cerebrovascular smooth muscle cation channels.

Evidence for a role of cation channels in vascular smooth muscle excitation. A major mechanism of vasoconstriction involves membrane potential depolarization and activation of voltage-dependent calcium channels (18). This leads to increased calcium entry and activation of calcium-dependent contractile mechanisms, such as enhancement of myosin light chain kinase activity (29). Although multiple ionic mechanisms of smooth muscle depolarization may be involved in this process, numerous studies have suggested a key role for cation channels in the depolarization of smooth muscle to various stimuli.

Comparison of DAG activated and TRP currents and their modulation. We have previously provided evidence that a mammalian homolog of a transient receptor protein (TRP) channel (TRPC6) is a key component of the cation currents activated by cell swelling and the depolarization of cerebrovascular smooth muscle to elevated intravascular pressure (25). Thus the PKC-activated cation currents and the pressure-induced depolarizing responses of intact arteries in the present study are likely mediated at least in part by the TRPC6 channel protein. It is interesting to note that cloned, expressed TRPC6 channels are activated by DAG, but this appears not to involve activation of PKC (7, 9). Thus DAG may be able to increase TRPC channel activity in a membrane-delimited fashion in some systems. This apparent lack of a role for PKC compared with the observations of the present study might be accounted for by differences in coupling of signaling pathways to TRPC channels in expression systems versus native cells. It is possible that heteromultimeric expression of TRPC channel proteins in native cells may dramatically alter the properties and signaling modalities of these channels, as has been reported when certain TRPC subtypes are coexpressed in various expression systems (13, 23).

Modulation of myogenic tone by PLC, DAG, and PKC. Numerous studies have demonstrated the involvement of G proteins, PLC, DAG, and PKC in the regulation of myogenic tone of intact arteries. Pertussis toxin inhibits myogenic tone of isolated cerebral arteries (21), indicating a role for G protein coupled events in the cell signaling associated with myogenic tone. PLC inhibitors typically eliminate myogenic tone (20). Narayanan and colleagues (16) found that PLC activity and DAG levels were increased in canine renal arteries when intravascular pressure was increased. Furthermore, PKC blockers inhibit myogenic tone in isolated cerebral arteries (20), skeletal muscle arterioles (5), and human coronary arteries (14) showing the general involvement of this kinase in the mechanisms underlying myogenic tone. In the present study we demonstrate that the depolarization of smooth muscle that occurs with increased intravascular pressure is linked to increased PKC activity. This suggests a specific mechanism by which PKC could regulate myogenic tone, i.e., through enhanced cation channel activity, which will increase cation influx and depolarize the vascular smooth muscle cells.

Physiological significance of present findings. The exact mechanisms of mechanotransduction initiating and sustaining myogenic tone remain unclear. However, mechanical stimulation of the smooth muscle cells in myogenically active arteries appears to activate G proteins coupled to PLC. Increased PLC activity leads to elevated DAG levels, which activates PKC. We propose that PKC then increases the activity of nonselective cation channels, depolarizing the smooth muscle membrane potential and enhancing calcium entry through voltage-dependent calcium channels. PKC activation is clearly associated with other signaling events that can lead to vasoconstriction, such as enhanced calcium sensitivity (8), and this response likely contributes to the overall mechanism of myogenic tone (3, 27). Simultaneous activation of multiple signaling events involving PKC could account, at least in part, for the high sensitivity of cerebral and other arteries that generate myogenic tone to changes in intravascular pressure over the physiological range of blood pressure.